Numerous methods are known for the assay of a target nucleic acid by using a nucleic acid probe. Many examples can be mentioned including those represented by (1) methods making use of a probe which utilizes the FRET (fluorescence resonance energy transfer) phenomenon (see, for example, Morrison et al., Anal. Biochem., 183, 231-244, 1989, and Xiangnin Chen et al., Proc. Natl. Acad. Sci. USA, 94, 10756-10761, 1977); (2) and methods making use of a probe which utilizes the characteristic of a fluorescence dye that the intensity of fluorescence emission is quenched as a result of its interaction with a particular nucleic acid base (see, for example, KURATA et al., Nucleic Acids Research, 29(6), e34, 2001). These methods measures a change or the amount of a change in an optical character (fluorescence intensity) of a fluorescence dye or the like, with which a nucleic acid probe is labeled, by hybridizing the labeled nucleic acid probe with a target nucleic acid and/or amplifying the target nucleic acid in a homogeneous system. Such a nucleic acid probe will hereinafter be called “a nucleic acid probe for a homogeneous solution system” throughout the specification. As an alternative, it may also be called simply “a nucleic acid probe” in some instances.
However, a nucleic acid probe for a homogeneous solution system, said probe being required in any one of the above-described methods, requires an oligonucleotide to be labeled with a fluorescence substance and/or a quencher substance. On top of this requirement, there is no standardized method for the designing of the probe. These circumstances have led to a waste of time and money. There is also an outstanding demand for further improvements in the assay sensitivity, although the assay sensitivity has been increasingly improved. Moreover, plural nucleic acids, which exist in a single system in the natural world and include unknown nucleic acids and/or known nucleic acids, cannot be assayed at the same time, simply and easily, specifically, accurately, in a short time, and with excellent sensitivity.
With the foregoing circumstances in view, the present invention has as an object to provide a novel method which makes it possible to assay at least one of unknown nucleic acids and/or known nucleic acids, which exist in a single system, simply and easily, specifically, accurately, in a short time, and with excellent sensitivity.